PROJECT SUMMARY Pachytene piRNAs are a population of small regulatory RNAs unique to mammals that regulates spermatogenesis and fertility. In mice, pachytene piRNAs are generated from long piRNA precursors through cleavage by the piRNA processing machinery and loaded onto two cytoplasmic PIWI proteins MIWI and MILI. However, the mechanism by which PIWI proteins are recruited to piRNA processing machinery to participate in piRNA biogenesis remains elusive. Here we provide preliminary data that reveal a novel genetic separation of MIWI and MILI to differentially enter into the piRNA processing machinery. This segregation is mediated by a mitochondria-anchored protein complex that specifically interacts with MIWI but not MILI. We hypothesize that distinct mitochondria- anchored complexes differentially direct different PIWI proteins into the piRNA pathway during pachytene piRNA processing. To test this hypothesis, we will use biochemical and mouse genetic approaches to: 1) understand the functional importance of specific mitochondria-based protein interactions in directing piRNA biogenesis and germ cell function; 2) define the coupling mechanism for PIWI protein recruitment and downstream piRNA processing; 3) explore a specific MILI recruiting mechanism to enter into the piRNA pathway. These studies will provide novel insight into the organizing principle of the piRNA processing machinery and the understanding of mechanism underlying pachytene piRNA biogenesis and its relevance to normal spermatogenesis and fertility.